Targeting the neuronal calcium sensor 1 for treating wolfram syndrome

ABSTRACT

The present invention relates to novel therapeutic ways for treating Wolfram Syndrome (WS) by targeting the neuronal calcium sensor 1 (NCS1). The present inventors have demonstrated that WFS1, which loss of function is responsible of the Wolfram Syndrome, forms a complex with the neuronal calcium sensor 1 (NCS1). The inventors have further demonstrated that WFS1 associates with NCS1 to prevent its degradation by the proteasome and that NCS1 regulates VDAC expression and mitochondrial respiratory chain. Thus, present invention provides an agonist of NCS1 for use in the treatment of WS. Such an agonist is e.g. a NCS1-encoding polynucleotide, an inhibitor of the proteasome or of calpains. The inventors have further shown that overexpression of NCS1 in WS cells allows increasing complex II driven respiration. The present invention further relates to a method for predicting the severity of WS by measuring the NCS1 level in a sample obtained from a patient.

FIELD OF THE INVENTION

The present invention relates to novel therapeutic ways for treatingWolfram Syndrome.

BACKGROUND OF THE INVENTION

Wolfram Syndrome (WS or DIDMOAD) is an autosomal recessiveneurodegenerative disorder characterized by diabetes insipidus (DI),diabetes mellitus (DM), optic atrophy (OA) and deafness (D). There aretwo types of Wolfram Syndrome, type 1 and type 2. The type 1 (WS1), themost common, is caused by a mutation in the WFS1 gene which encodeswolframin, a transmembrane protein located in the endoplasmic reticulum(ER). Wolframin plays a role in ER calcium homeostasis and in severalprotein responses. This protein is abundantly expressed in the pancreas,brain, heart and muscle (Rigoli. L and Di Bella. C, Curr Opin Pediatr.2012; 24(4):512-7). Mutations in the WFS1 gene leads to apartially/totally inactivated wolframin which results in the developmentof endoplasmic reticulum stress, leading to apoptosis.

The morbidity and mortality in WS1 are very high and the median age ofdeath is around 30. WS1 usually occurs in the first decade of life withthe apparition of a diabetes mellitus type 1 and an optic atrophy. 50%of patients also develop diabetes insipidus and present some degree ofdeafness. Around 65% of the patients develop the whole four DIDMOADsymptoms. Some patients additionally present neurological abnormalities(62% of the patients) such as ataxia of the trunk, urinary tractabnormalities (urinary incontinence, recurrent infections, hydroureter)gastrointestinal tract affections (such as bowel dismotility) andprimary hypogonadism. Around 60% of the patients are affected by severeepisodes of depression, psychosis, or organic brain syndrome as well asimpulsive verbal and physical aggression. Magnetic resonance imagingscans demonstrate generalized brain atrophy, especially in thecerebellum, medulla, and pons; absence of signal from the posteriorpituitary; and reduced signal from the optic nerve (Ito S et al., AJNRAm J Neuroradiol, 2007; 28:305-306). Central apnea, due to bulbardysfunction is a common cause of mortality in WS1 patients.

No treatment of Wolfram Syndrome is known to date. The managementconsists in treating the symptoms e.g. with insulin replacement and acontrolled diet to treat diabetes mellitus, prophylactic antibiotherapyfor preventing recurrent urinary tract infections.

Thus, there is a real need to develop novel and efficient therapieswhich would allow treating Wolfram Syndrome or at least reducing itssymptoms.

DETAILED DESCRIPTION

The present inventors have discovered that WFS1, which loss of functionis responsible of the Wolfram Syndrome, forms a complex with theneuronal calcium sensor 1 (NCS1) and further with inositol1,4,5-triphosphate receptor 1 (ITPR1) and voltage-dependent anionchannel 1 (VDAC1) to promote ER-mitochondrial Ca2+ transfer. Theinventors have further demonstrated that WFS1 associates with NCS1 toprevent its degradation by the proteasome and that NCS1 regulates VDACexpression and mitochondrial respiratory chain.

As used herein, the expression “NCS1 encoding gene” refers to theneuronal calcium sensor 1 gene of any species to which the methodsaccording to the invention can apply. Particularly, the NCS1 encodinggene is human. The human NCS1-encoding gene is also known as the “FREQgene” localized on chromosome 9 at position 9q34.11 and is quiteconserved between species (Bourne et al., Journal of BiologicalChemistry, 276, 11949-11955). NCS1 is an EF-hand cytosolic proteinpreferentially expressed in neurons (Pongs et al., Neuron, 1993, 11,15-28) and known to regulate inositol 1,4,5-triphosphate receptor (ITPR)(Nakao et al. PloS one 2015, 10,e0125050; Schlecker et al., The Journalof clinical investigation, 2006, 116, 1668-1674; Zhang et al.,Experimental eye research, 2014, 125, 30-40) and Dopamine D2 receptor(Kabbani et al., J. Neurosci, 2002, 22, 8476-8486).

The expression “NCS1-encoding gene” should be understood broadly. In thecontext of the present invention, the NCS1-encoding gene can have theexact same sequence as the above-mentioned FREQ gene or can be anyfunctional variant thereof. A functional variant of FREQ is a sequencethat will produce a functional NCS1 protein. The skilled person knowshow to select FREQ functional variants suitable for the purpose of thepresent invention. In the context of the present invention, a“functional NCS1 protein” means that NCS1 is capable of binding ITPR1 topromote ER-mitochondrial Ca2+ transfer. Thus, a suitable test fordetermining if NCS1 is “functional” in the context of the presentinvention consists in evaluating if said NCS1 is able 1/ to bind toITPR1 and 2/ to promote the Ca2+ mitochondria uptake. A suitable testfor detecting the NCS1/ITPR1 binding and the NCS1/ITPR1-mediatedmitochondria Ca2+ uptake is disclosed in Example 1 of the pendingapplication.

Typically, variants of the FREQ gene according to the invention presenta sequence identity of at least 70%, 75%, 80% or more particularly 90%with the FREQ sequence.

As used herein, the percentage of sequence identity refers tocomparisons between nucleic acid sequences, and is determined bycomparing two optimally aligned sequences over a comparison window,wherein the portion of the nucleic acid sequence in the comparisonwindow may comprise additions, deletions (i.e., gaps), or substitutionsas compared to the reference sequence (which does not compriseadditions, deletions or substitutions) for optimal alignment of the twosequences. The skilled person will know how to determine the percentageof identity between two nucleic acid sequences.

The “WFS1 gene” (gene bank ID: 7466) codes for wolframin, a predicted890-amino acid transmembrane protein. It is located at position 4p16.1in the human genome and its mutation(s) is(are) responsible of theWolfram Syndrome of type 1. The following mutations of WFS1, which areidentified by their rs reference in the NCBI dbSNP Short GeneticVariations Database, are examples of mutations known to cause WolframSyndrome 1: rs28937890, rs28937891, rs104893879, rs28937892,rs104893880, rs104893881, rs587776598, rs71524377. Most of the mutationsassociated with Wolfram syndrome are spread over the entire codingregion and are typically inactivating (Cryns et al., Hum. Mutat., 2003,22: 275-287).

In the context of the invention, the term “treating” or “treatment”,means reversing, alleviating, inhibiting the progress of, or preventingthe disorder or condition to which such term applies, or one or moresymptoms of such disorder or condition. A “therapeutically effectiveamount” is intended for a minimal amount of active agent (e.g.,NCS1-encoding polynucleotide) which is necessary to impart therapeuticbenefit to a subject. For example, a “therapeutically effective amount”to a mammal is such an amount which induces, ameliorates or otherwisecauses an improvement in the pathological symptoms, disease progressionor physiological conditions associated with or resistance to succumbingto a disorder.

As used herein, the term “subject” denotes a mammal, particularly ahuman.

Treatments of the Wolfram Syndrome According to the Invention

In a first aspect, the inventors have demonstrated that WFS1 interactswith NCS1 and modulates its expression. They have observed that theprotein level of NCS1 was significantly reduced (of around 50%) inWolfram patients cells due to an over-degradation of NCS1 by theproteasome. By interacting with NCS1, WFS1 also interacts with ITPR1,and thereby regulates the mitochondrial Ca2+ uptake.

Ca2+ is an important actor in several pathways of the mitochondrialfunction, particularly in the respiratory complexes. Patient cellspresented a significant decrease (around 20%) in the expression of thecomplex II (SDHA) and complex III (UQCRC2) subunits. Accordingly,Wolfram syndrome induces a loss of mitochondrial proteins, therebyimpairing the mitochondrial function.

The inventors have demonstrated that the overexpression of NCS1 is ableto significantly increase the complex II respiration in Wolframpatient's cells, and is thus able to counter some effects of the WFS1mutation.

Increasing/stimulating the expression of NCS1 is thus a very efficientpathway for treating Wolfram Syndrome.

Accordingly, in a first aspect, the present invention relates to anagonist of NCS1 for use in the treatment of Wolfram Syndrome.

An “agonist” of NCS1 should be understood broadly. In the context of thepresent invention an agonist is a compound which positively modulatesthe expression/activity of NCS1. Such a compound can be e.g.:

an agent stimulating the expression of NCS1 (methods for determiningwhether a compound increases or activates the expression of NCS1 aree.g. disclosed in U.S. Pat. No. 7,230,155);an agent preventing the degradation of NCS1;an agent mimicking NCS1's expression/activity; oran agent stabilizing NCS1 interaction with WFS1; thereby preventing itroutine to the proteasome.

In the context of the present invention, the effects of the agonist canbe evaluated by measuring changes in the NCS-1 quantity, or by measuringthe downstream effects of NCS-1 function, for example by measuring thecomplex II respiration.

Compounds Stimulating the Expression of NCS1

NCS1-Encoding Polynucleotide

In a first embodiment, the “agonist” according to the present inventionis a NCS1-encoding polypeptide.

Thus, the present invention relates to a NCS1-encoding polynucleotidefor use in the treatment of Wolfram Syndrome.

The present invention also provides a method for treating WolframSyndrome comprising administering, to a patient in need thereof, aNCS1-encoding polynucleotide

Gene therapy is a particularly convenient way to treat Wolfram Syndromeas it enables the provision of an additional NCS1 polypeptide, forexample as discussed below.

Gene therapy may be carried out by means of supplementation of cellslacking a functional NCS1 polypeptide with a functional NCS1. Productionof a suitable gene product may be achieved using recombinant techniques.For example, a suitable vector may be inserted into a host cell andexpressed in that cell.

Thus, the invention relates to a method for treating Wolfram Syndromewhich comprises the step of administering in a subject in need thereof aNCS1-encoding polynucleotide, i.e. a nucleic acid sequence that encodesa functional NCS1, so that NCS1 is expressed in vivo by the cells of thesubject that have been transfected with said polynucleotide.Accordingly, said method leads to an overexpression of wild-type NCS1which compensates the deregulated over-degradation of NCS1 by theproteasome.

The invention also relates to the use of a NCS1-encoding polynucleotidefor the manufacture of a medicament intended for the treatment ofWolfram Syndrome.

Said NCS1-encoding polynucleotide is administered in a therapeuticallyeffective amount.

Preferably the NCS1 sequence according to the invention is associatedwith elements that enable for regulation of its expression, such as apromoter sequence.

Such a nucleic acid may be in the form of a vector. As used herein, theterm “vector” refers to a nucleic acid molecule capable of transportinganother nucleic acid to which it has been linked. One type of vector isa “plasmid”, which refers to a circular double stranded DNA loop intowhich additional DNA segments can be ligated. Another type of vector isa viral vector, wherein additional DNA segments can be ligated into theviral genome. Certain vectors are capable of autonomous replication in ahost cell into which they are introduced (e.g., bacterial vectors havinga bacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors,expression vectors, are capable of directing the expression of genes towhich they are operably linked. In general, expression vectors ofutility in recombinant DNA techniques are often in the form of plasmids(vectors). However, the invention is intended to include such otherforms of expression vectors, such as viral vectors (e.g., replicationdefective retroviruses, adenoviruses, lentiviruses and adeno-associatedviruses (AAV)), which serve equivalent functions. Adeno-associated viralvectors have proven to be very useful for transferring gene-encodingpolynucleotides to several tissues, such as retina (see Surace et al.,Vision Serach 48, 353-359, 2008; Hellström et al., Gene Therapy, 16,521-532, 2009; Cwerman-Thibault et al., Molecular Therapy-Methods &Clinical Development 2, 15003, 2015 or Bemelmans et al., PLoS ONE 8(4):e61618, 2013). Thus, in the context of the present invention, AAVvectors represent very promising tool for directly delivering theNCS1-encoding polynucleotide to tissues affected during WS, e.g. to theretina.

Accordingly, in a preferred embodiment, the viral vector is anadeno-associated virus vector.

Different types of AAV vectors can be used depending on the type ofadministration and on the tissue to be targeted. For instance, the AAV2vector has proven to be particularly efficient when administered locally(e.g. by intra-ocular administration for targeting retina: see Surace etal, 2008; Hellström et al., 2009; and Cwerman-Thibault et al., 2015),whereas the scAAV9 and 10 vectors are particularly efficient whenadministered systemically (Bemelmans et al, 2013).

The NCS1-encoding polynucleotide may be introduced into a target cell bymeans of any procedure known for the delivery of nucleic acids to thenucleus of cells, ex vivo, on cells in culture or removed from an animalor a patient, or in vivo.

Ex vivo introduction may be performed by any standard method well knownby one skilled in the art, e.g. transfection, electroporation,microinjection, transduction, cell fusion, DEAE dextran, calciumphosphate precipitation, or use of a gene gun.

The NCS1-encoding polynucleotide can also be introduced ex vivo or invivo by lipofection. In certain embodiments, the use of liposomes and/ornanoparticles is contemplated for the introduction of the donor nucleicacid targeting system into host cells.

Nanocapsules can generally entrap compounds in a stable and reproducibleway. To avoid side effects due to intracellular polymeric overloading,such ultrafine particles (sized around 0.1 μm) should be designed usingpolymers able to be degraded in vivo. Biodegradablepolyalkyl-cyanoacrylate nanoparticles that meet these requirements arecontemplated for use in the present invention, and such particles may beare easily made.

Liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MLVs)). MLVs generally havediameters of from 25 nm to 4 μm. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200 to 500 Å, containing an aqueous solution in the core.

Synthetic cationic lipids designed to limit the difficulties and dangersencountered with liposome mediated transfection can be used to prepareliposomes for in vivo transfection of a gene encoding a marker. The useof cationic lipids may promote encapsulation of negatively chargednucleic acids, and also promote fusion with negatively charged cellmembranes (Felgner et al., 1989).

Alternatively, one of the simplest and the safest way to deliver theNCS1-encoding polynucleotide across cell membranes in vivo may involvethe direct application of high concentration free or nakedpolynucleotides (typically mRNA or DNA). By “naked DNA (or RNA)” ismeant a DNA (RNA) molecule which has not been previously complexed withother chemical moieties. Naked DNA uptake by animal cells may beincreased by administering the cells simultaneously with excipients andthe nucleic acid. Such excipients are reagents that enhance or increasepenetration of the DNA across cellular membranes and thus delivery tothe cells delivery of the therapeutic agent. Various excipients havebeen described in the art, such as surfactants, e.g. a surfactantselected form the group consisting of Triton X-100, sodium dodecylsulfate, Tween 20, and Tween 80; bacterial toxins, for instancestreptolysin 0, cholera toxin, and recombinant modified labile toxin ofE coli; and polysaccharides, such as glucose, sucrose, fructose, ormaltose, for instance, which act by disrupting the osmotic pressure inthe vicinity of the cell membrane. Other methods have been described toenhance delivery of free polynucleotides, such as blocking ofpolynucleotide inactivation via endo- or exonucleolytic cleavage by bothextra- and intracellular nucleases.

Knowing the sequence of the NCS1 gene (FREQ), one skilled in the art canreadily produce said polypeptides, by standard techniques for productionof polypeptides. For instance, they can be synthesized using well-knownsolid phase method, preferably using a commercially available peptidesynthesis apparatus (such as that made by Applied Biosystems, FosterCity, Calif.) and following the manufacturer's instructions.

Alternatively, the polypeptides of the invention can be synthesized byrecombinant DNA techniques as is now well-known in the art. For example,these fragments can be obtained as DNA expression products afterincorporation of DNA sequences encoding the desired (poly)peptide intoexpression vectors and introduction of such vectors into suitableeukaryotic or prokaryotic hosts that will express the desiredpolypeptide, from which they can be later isolated using well-knowntechniques.

Polypeptides of the invention can be use in an isolated (e.g., purified)form or contained in a vector, such as a membrane or lipid vesicle (e.g.a liposome).

Activators of NCS1's Expression

In another embodiment, the NCS1 agonist is a compound which induces anactivation/increase in the expression of the NCS1 gene (FREQ).

Such compounds can be chemical molecules or proteins that bind to themRNA of a gene encoding a NCS-1 polypeptide, thereby stabilizing thenative conformation of the mRNA and facilitating transcription and/ortranslation.

Calcium was shown to increase the expression of NCS1 (seeHamasaki-Katagiri et al, J Biol Chem. 2010 Feb. 12; 285(7):4405-14).This increase is prevented by FK506, a calcineurin inhibitor.

Inhibitors of NCS1+s Repression

In a further embodiment, the agonist according to the present inventioncan inhibit NCS1's repression, i.e. inhibit a repressor (inhibitor) ofNCS1 expression.

Thus, in a particular embodiment, the NCS1 agonist according to thepresent invention inhibits the repressors of NCS1.

In this case, the NCS1's agonist may be based on anti-senseoligonucleotide constructs. Anti-sense oligonucleotides, includinganti-sense RNA molecules and anti-sense DNA molecules, would act todirectly block the translation of the inhibitor of NCS1 by bindingthereto and thus preventing protein translation or increasing mRNAdegradation, thus decreasing the level of the target inhibitor, and thusits NCS1 inhibiting activity. Methods for using antisense techniques forspecifically inhibiting gene expression of genes whose sequence is knownare well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131;6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732).

Small inhibitory RNAs (siRNAs) can also inhibit NCS1's inhibitorsexpression. In this case, their expression is reduced by contacting asubject or cell with a small double stranded RNA (dsRNA), or a vector orconstruct causing the production of a small double stranded RNA, suchthat their expression is specifically inhibited (i.e. RNA interferenceor RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encodingvector are well known in the art for genes whose sequence is known (e.g.see Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, M T. et al. (2002); Brummelkamp, T R. et al. (2002);U.S. Pat. Nos. 6,573,099 and 6,506,559; and International PatentPublication Nos. WO 01/36646, WO 99/32619, and WO 01/68836).

Compounds Preventing NCS1's Degradation

As disclosed above, WFS1 interacts with NCS1 and the NCS1 level ishighly affected in Wolfram-patients cells. WFS1 regulates NCS1 proteinstability by protecting NCS1 from proteasomal degradation.

Thus, preventing the degradation of NCS1 can also represent a promisingtool for treating Wolfram Syndrome.

Inhibitors of the Proteasome

The inventors have demonstrated that it is possible to increase the NCS1level in patient cells by using an inhibitor of the proteasome.Accordingly, inhibiting the proteasome pathway in Wolfram patients is avery efficient way for treating Wolfram syndrome.

Thus, in another aspect, the agonist of NCS1 according to the presentinvention is an inhibitor of the proteasome.

By an “inhibitor of the proteasome”, it is herein referred to a compoundwhich has the ability to reduce or prevent the degradation of proteinsby the proteasome.

As disclosed in Grigoreva et al. (Grigoreva et al., Oncotarget, 2015,1-17), several pathways can be targeted for inhibiting the proteasomeactivity. The proteasome-degradation system involves different actors:proteins which are to be degraded by the proteasome are firstlyubiquitinated by the E1-E2-E3 ubiquitination enzymatic cascade. Onceubiquitinated, the proteins are then transferred to the proteasome fordegradation. The proteasome, also kwon as 26S proteasome, is amulti-unit enzyme complex consisting in the association of a cylindrical20S complex with one or two regulatory 19S complex(es). The 19S complexprepares the substrate protein for degradation in the 20S complex.

Accordingly, inhibitors of the proteasome can either target theubiquitination pathway or the proteasome structure/activity itself (seeGrigoreva et al; Pellom et al, Clin. Cell Immunol., 2012, S5; and Liu etal, Biochimica et Biophysica Acta 1855, 2015, 50-60).

Inhibitors of the proteasome are well known by the skilled person (seee.g. Nalepa et al., Nature Reviews, 2006, 5596-613; Bedford et al.,Nature Reviews, 2010, 10, 29-46; Liu et al., Biochimica et BiophysicaActa 1855, 2015, 50-60; Pellom Jr. et al., Clin. Cell Immunol., 2012, S5and Grigoreva et al., Oncotarget, 2015, 1-17).

The following list discloses a non-exhaustive number of known inhibitorsof the proteasomic activity.

Metcalf et al (Expert Opinion on Therapeutic Patents, 2014, 24:4,369-382) and Pevzner et al (Expert Opinion on Drug Discovery, 2013, 8:5,537-568) review all the major proteasome inhibitors known to date.

Boronic-acid based inhibitors: Peptide boronates selectively target theproteasome. They bound with the N-terminal threonine residue of theproteasome by a non-covalent bound.

Examples are:

-   -   Bortezomib. (See e.g. Luker et al., Nature medicine, 2003, 9(7),        969-973 and Adams et al., Cancer investigation, 2004, 22(2),        304-311). It is one of the most well-known proteasome        inhibitors;    -   Delanzomib (CEP-18770) (See e.g. Berkerset al., Molecular        pharmaceutics, 2012, 9.5: 1126-1135);    -   Ixazomib (MLN9708) (See Kupperman et al., Cancer Research, 2010,        70:1970-1980).        Peptide epoxyketone: They contain an α,β-epoxyketone moiety that        assists in the formation of of a morpholino adduct with the        N-terminal threonine residue of the proteasome.

Examples are:

-   -   Carfilzomib (PR-171) (See J Kuhn et al., Current cancer drug        targets, 2001, 11(3):285-295);    -   Oprozomib (ONX-0912) (See Hurchla et al., Leukemia, 2013, 27(2),        430-440);    -   Epoxomicin (See Hanada et al., J. Antibiot. 1992, 45,        1746-1752).        Peptide aldehydes: They act against serine and cysteine protease        and bind to the proteasome via nucleophilic binding.

Examples are:

-   -   MG132 (See for instance Momose et al., Bioscience, Biotechnology        and Biochemistry, 2005, 69:1733-1742 and Alexandrova et al.,        Cell biochemistry and function, 2008, 26(3):392-398);    -   Tyropeptin A (See e.g. Momose et al., 2005; Hines et al.,        Chemistry and Biology, 2008, 15:501-512 and Momose et al.,        Bioscience, Biotechnology and Biochemistry, 2002, 66:2256-2258);    -   Felutamide B (See e.g. Hines et al., Chemistry and Biology,        2008, 15:501-512).        Salinosporamide analogs: they inhibit the catalytic activity of        the 20S subunit by forming a complex with it.

Examples are:

-   -   Salinosporamide A (Marizomib): (see Macherla et al., J. Med.        Chem., 2005, 48:3684-3687).        β-lactones:

Examples are:

-   -   Omuralide/clasto-lactacystin-βlactones: creates a covalent bound        with the catalytic Thr1 of the 135 subunit of the proteasome.        (See Smith et al., Molecular Medecine, 2002, 8:382-392 and        Hasegawa et al., Bioorganic & MedicinalChemistry letters, 2008,        18:5668-5671).        Tea polyphenols and polyphenol derivatives such as the Flavonoid        compound Epigallocatechin gallate, possess the ability to bind        to the proteasome (See Nam et al., J. Biol. Chem,. 2001,        276:13322-13330).        Belactosin: This compound is a Streptomyces metabolite which has        the ability to efficiently inhibit the 20S subunit activity (see        Asai et al., Biochem. Pharmacol. 2004, 67, 227-234).        Tyropeptins such as TP-110, a tyropeptin A derivative,        specifically inhibits the chymotrypsin-like activity of the 20S        mammalian proteasome (See Momose et al., Bioscience,        Biotechnology and Biochemistry, 2005, 69:1733-1742).        Syrbactins such as syringolins and glidobactins are considered        as promising proteasome inhibitors (see Clerc et al., Proc Nat        Acad Sci, 2009, 106(16), 6507-6512).        Vinyl Sulfone and vinyl ester-containing compounds: they inhibit        the proteasome by forming a covalent bound with the catalytic        THR1 residues of the proteosomal active sites (see Baldisserotto        et al., Bioorg Med Chem Lett, 2009, 19(7), 1966-1969).        TMC-95A is a cyclic tripeptide which inhibits all three        proteolytic activities of the proteasome by a non-covalent link        (see Koguchi et al., Chem Inform, 2000, 31(26)).        Metal complexes containing gold, zinc, nickel or copper have        also been shown to have proteasome-inhibiting properties (see        e.g. Milacic et al., Cancer Res, 2006, 66(21), 10478-86; Cvek et        al., J. Med Chem, 2008, 51(20), 6256-8 and Daniel et al., Breast        Cancer Res, 2005, 7(6), R897-908).

As disclosed above, several inhibitors of the proteasome activity or ofthe ubiquitination pathway are known to date. The skilled person willknow how to select a suitable inhibitor for the purpose of the presentinvention.

In a further aspect, the present invention relates to a method fortreating Wolfram Syndrome comprising administering, to a patient in needthereof, a therapeutically effective amount of an inhibitor of theproteasome.

Calpain Inhibitors

Several studies have reported that NCS1 is also degraded by calpains(see e.g. Benbow et al. The Journal of Biological Chemistry, vol 286,40, 34575-34582, 2011; Blachford et al., Cell Calcium, vol 46, 257-262,2009 and Boehmerle et al, PNAS, vol. 104,26, 11103-11108, 2007).Accordingly, inhibiting the calpain-mediated degradation of NCS1 alsorepresents a promising way for treating WS, as it would help enhancingthe NCS1 level in affected cells.

Thus, in a further aspect, the agonist of NCS1 according to the presentinvention is a calpain inhibitor.

Calpains are calcium-dependent non-lysosomal neutral cysteine proteases.As disclosed above, they have the ability to specifically target andcleave NCS1, thereby preventing its interaction with ITPR1 and the Ca²⁺intake resulting thereof. Boehmerle (Boehmerle, 2007), Blachford(Blachford, 2009) and Benbow (Benbow, 2011), have all demonstrated thatcalpain inhibition allowed recovering a physiological intra-cellularlevel of NCS1, thereby allowing the recovery of the ITPR1-Ca²⁺ mediatedsignaling.

In the context of the present invention, “a calpain inhibitor” is acompound which has the ability to reduce or prevent the degradation ofNCS1 by calpains.

The skilled person knows several compounds having this type of activity.Calpain inhibitors have already been disclosed for the treatment ofvarious pathologies such as inflammatory bowel diseases (see e.g.WO2015/066212, WO2012001121, JP2013063953; JP2010006834; CN104083350). ODonkor et al (Expert Opinion on Therapeutic Patents, 21:5, 601-636, 2011and Expert Opinion on Therapeutic Patents, 25:1, 17-31, 2015) and Sikloset al (Acta Parmaceutica Sinica B, 5:6, 506-519, 2015) review all themajor calpain inhibitors known to date.

Among these compounds, one may cite the following compounds:

Non-peptide calpain inhibitors such as carboxamides (Lubisch et al.,Bioorg Med Chem Lett., 10(19), 2187-91, 2000; Lee et al, Eur J MedChem., 44(3), 1331-4, 2009; Kim et al., Eur J Med Chem., 46(5), 1721-8,2011), Dihydroxychalcones (Baek et al., Bioorg Chem., 51, 24-30, 2013);α-mercaptoacrylates (Rasbach et al., J Med Chem. 8, 52(1), 181-188,2009).Peptidomimetic calpain inhibitors such as epoxysuccinate-basedinhibitors (Schiefer et al., J Med Chem.; 56(15): 6054-6068, 2013),α-Helical inhibitors (Jo et al., J Am Chem Soc., 24, 134(42), 17704-13,2012), peptidomimetic macrocycles (Chen et al., Chem Biodivers., 9(11),2473-84, 2012; Abell et al., Angew Chem Int Ed Engl. 48(8),1455-8,2009).

The inhibitor of the proteasome or of calpains according to theinvention can be administered by any suitable route of administration.For example, the inhibitor according to the invention can beadministered by oral (including buccal and sublingual) or parenteral(including intramuscular, intra-arterial, intrathecal, subcutaneous andintravenous) administration. The skilled person will know which dosageand route of administration are the most adapted depending on the typeof inhibitor selected.

In a further aspect, the present invention relates to a method fortreating Wolfram Syndrome comprising administering, to a patient in needthereof, a therapeutically effective amount of an inhibitor of calpains.

Compounds Mimicking NCS1 Expression/Activity

As disclosed above, during WS, the over-degradation of NCS1 induces asevere impairment in the mitochondrial respiratory chain. Thus, withoutbeing bound by theory, the inventors believe that reproducing a NCS1activity, e.g. by using compounds mimicking NCS1 activity, represents aninteresting treatment pathway for treating WS.

NCS1 Peptidomimetics

Thus, in a further embodiment, the agonist according to the presentinvention is a NCS1 peptidomimetic (see e.g. Vagner et al, Curr OpinChem Biol. 2008 Jun. 12(3): 292-296). The skilled person knows severaltechniques allowing the design and synthesis of peptidomimetics. Mimeticanalogs of the NCS-1 polypeptide or biologically active fragmentsthereof can be generated by, for example, by substituting the aminoacids that are expected to be essential for the biological activitywith, e.g., stereoisomers, i.e. D-amino acids (see e.g., Tsukida, J.Med. Chem. 40 (1997), 3534-3541). The structure-based design andsynthesis of low-molecular-weight synthetic molecules that mimic theactivity of native biological polypeptide is further described in, e.g.,Dowd, Nature Biotechnol. 16 (1998), 190-195; Kieber-Emmons, CurrentOpinion Biotechnol. 8 (1997), 435-441; Moore, Proc. West Pharmacol. Soc.40 (1997), 115-119; Mathews, Proc. West Pharmacol. Soc. 40 (1997),121-125; Mukhija, European J. Biochem. 254 (1998), 433-438.

Compounds Stabilizing the NCS1/WFS1 Interaction

As previously disclosed, the inventors have demonstrated that WFS1interacts with NCS1 thereby preventing the degradation of NCS1 by theproteasome. Thus, stabilizing the NCS1/WFS1 interaction can alsorepresent a very interesting way for preventing the degradation of NCS1during WS.

Thus, in a further embodiment, the agonist according to the presentinvention is a compound which stabilizes the WFS1/NCS1 interaction. Anexample of stabilizer is Lithium.

Methods for Predicting the Severity of Wolfram Syndrome in a Patient

In a further embodiment, the present invention relates to a method forassessing the severity of Wolfram syndrome in a subject comprising thestep of measuring the level of NCS1 in a biological sample obtained fromsaid subject, wherein the level of NCS1 is negatively correlated withthe severity of WS.

The present inventors have indeed established a correlation between theexpression level of NCS1 and the severity of WS.

The term “biological sample” means any biological sample derived from asubject. Examples of such samples include fluids, tissues, cell samples,organs, biopsies, etc. Preferred biological samples are a cell or tissuesample. Typically, the biological sample is a fibroblast sample.

The “NCS1 level” in a sample refers to the concentration of NCS1 in saidsample.

Typically, the level of NCS1 measured in the sample obtained from apatient is compared to a control value. Such control value may bedetermined in regard to the level of NCS1 present in samples taken fromone or more healthy subject or to the NCS1 distribution in a controlpopulation.

Typically, the lower the level of NCS1 is, the more severe Wolframsyndrome is.

The severity of the WS can be scaled e.g. according to the WolframUnified Rating Scale (WURS) (see Nguyen et al, Orphanet J Rare Dis.,7,89, 2012).

In a particular embodiment, the method for determining if a subject ispredisposed to having severe Wolfram syndrome according to the inventionis performed after a step of determining whether a patient has WS ornot. WS can be diagnosed by any known method by the skilled person, e.g.by detecting one of the particular mutations of WFS1 disclosed above(see e.g. Strom et al., Hum. Molec. Genet, 1998, 7, 2021-2028).

Determination of the NCS1 level can be performed by a variety of methodsknown by the skilled person for determining the protein level in asample.

Such methods comprise contacting the sample with a binding partnercapable of selectively interacting with the NCS1 present in the sample.The binding partner is generally an antibody that may be polyclonal ormonoclonal, preferably monoclonal.

The presence of the protein can be detected using standardelectrophoretic and immunodiagnostic techniques, including immunoassayssuch as competition, direct reaction, or sandwich type assays. Suchassays include, but are not limited to, Western blots; agglutinationtests; enzyme-labeled and mediated immunoassays, such as ELISAs;biotin/avidin type assays; radioimmunoassays; immunoelectrophoresis;immunoprecipitation, etc. The reactions generally include revealinglabels such as fluorescent, chemiluminescent, radioactive, enzymaticlabels or dye molecules, or other methods for detecting the formation ofa complex between the antigen and the antibody or antibodies reactedtherewith.

ELISA method can be used, wherein the wells of a microtiter plate arecoated with an anti-NCS1 antibody. A biological sample to be tested isthen added to the coated wells. After a period of incubation sufficientto allow the formation of antibody-antigen complexes, the plate can bewashed to remove unbound moieties and a detectably labelled secondarybinding molecule added. The secondary binding molecule is allowed toreact with any captured sample marker protein, the plate washed and thepresence of the secondary binding molecule detected using methods wellknown in the art.

The present invention will be further disclosed in the followingexamples.

BRIEF DESCRIPTION OF THE FIGURES

Figure Legends

FIG. 1: WFS1 interacts with and modulates the expression of NCS1

(A) Co-immunoprecipitation (IP) of Wfs1-myc with Ncs1-Flag with antibodyagainst Flag from lysates of transfected HEK293T.

Immunoprecipitates were analysed by western blotting with antibodiesagainst Flag and myc. Input represents cell lysate. n=3 independentexperiments. (B) Transcript levels of WFS1 were quantified by RT-qPCR incontrol (C1, C2) and patient fibroblasts (P1, P2). The mRNA levels werenormalized with reference gene L27. Error bars: SEM n=6 independentexperiments. (C) Immunoblot of lysates from controls (C1, C2) andpatient fibroblasts (P1, P2) with NCS1 antibody. GAPDH was used as aloading control. The immunoblot bands were quantified by densitometry,and the NCS1/GAPDH ratios were calculated (n=5 independent experiments).(D) Western analysis and quantification of control fibroblaststransfected with scramble siRNA (siScr) or WFS1 siRNA (siWFS1). WFS1 andNCS1 protein expression were analysed 72 h post-siRNA transfection. Dataare represented as mean ±SEM from n=7 independent experiments *p<0.05,using Mann Whitney test. (E) Control and patient cells were treated with10 μM of MG-132, followed by time course immunoblotting of NCS1.

GAPDH was used as a loading control. Quantification of NCS1 expressionlevel after 16 h of MG-132 treatment normalized to GAPDH is shown. Dataare represented as mean ±SEM from n=3 independent experiments *p<0.05.

FIG. 2: WFS1 regulates mitochondrial functionality

(A) Western blot of NDUFA13 (complex I), SDHA (complex II) and UQCRC2(complex III) (left) and densitometric analysis (right) in control andpatient fibroblasts. GAPDH was used as a loading control. (mean ±SEM,n=6) ***p<0.005 vs. control. (B) Western blot of MTCO1 (complex IV) andATPsynthase (complex V) (left) and densitometric analysis (right) incontrol and patient fibroblasts. GAPDH was used as a loading control.(mean ±SEM, n=4). (C) Mitochondrial complex I and complex II-dependentrespiratory rate. Respiratory rate: nmol oxygen consumed/min/mg protein.(mean ±SEM, n=5 experiments, *p<0.05, ***p<0.005). (D) Complex I, II andIII enzymatic activities in control and patient fibroblasts. (mean ±SEM,n=3 experiments, *p<0.05).

FIG. 3: NCS1 regulates VDAC1 expression and mitochondrial respiratorychain.

(A) Complex I and complex II mediated respiration of control fibroblaststransfected with non-targeting siRNA (siScr) or WFS1 siRNA (siWFS1)(bottom). (mean ±SEM, n=7 experiments, *p<0.05). (B) Complex I andcomplex II-mediated respiration of control fibroblasts transfected withnon-targeting siRNA (siScr) or NCS1 siRNA (siNCS1) (bottom). (mean ±SEM,n=7, *p<0.05). (C) Complex I and complex II-mediated respiration ofcontrol fibroblasts transfected with nontargeting siRNA (siScr) or NCS1siRNA (siNCS1) and WFS1 siRNA (siWFS1) (bottom). (mean ±SEM, n=3,*p<0.05). (D) Effect of NCS1 and WFS1 knockdown on NCS1, VDAC1 and SDHA(complex II) protein expression in control fibroblasts transfected withnontargeting (siScr), NCS1 (siNCS1) or WFS1 (siWFS1) siRNA. NCS1knockdown effect is shown 72 h and 96 h after siRNA transfection. GAPDHwas used as a control of specificity and loading. The immunoblot bandswere quantified by densitometry, and the NCS1, VDAC1 and SDHA intensitywere calculated (mean ±SEM, n=7 experiments, *p<0.05, **p<0.01).

FIG. 4: Overexpression of NCS1 in fibroblasts:

(a) NCS1 expression level in control (C) and WFS1mutant cells (P). (b)represents complex 1 and II respiration rates in controls (C) and inWFS1 mutant cells (P). (c) Overexpression of NCS1 in mutant cells. (d)Represents the complex 1 and II respiration rates in mutant cellstransfected either with NCS-1-Flag or with Flag alone.

EXAMPLES Example 1: The Endoplasmic Reticulum-Mitochondria Crosstalk isRegulated by WFS1/NCS1 Interaction and is Impaired in Wolfram SyndromeSummary

Communication between endoplasmic reticulum (ER) and mitochondria playsa pivotal role in calcium (Ca2+) signaling, energy metabolism and cellsurvival. Dysfunctions of this crosstalk lead to metabolic andneurodegenerative diseases. Wolfram syndrome is a fatalneurodegenerative disease due to mutations of the ER resident proteinWFS1. However, clinical phenotype of WS resemble mitochondrialdisorders. Here we show that WFS1 forms a complex with NCS1, inositol1,4,5-triphosphate receptor (ITPR) and voltage-dependent anion channel 1(VDAC1) to promote ER-mitochondrial Ca2+ transfer. Moreover, wedemonstrate that WFS1 associates with NCS1 to prevent its degradation bythe proteasome. Finally, we show that NCS1 regulates VDAC expression andmitochondrial respiratory chain. Our results describe an unexpected keyrole of WFS1 and NCS1 in ER-mitochondria crosstalk and reconcile the ERexpression of WFS1 with the mitochondrial phenotype, underlining a novelpathogenic mechanism for WS and opening new insights into the biogenesisof other neurodegenerative diseases.

Introduction

Mitochondria exert essential cellular functions, from bioenergetics andmetabolism to ion homeostasis and apoptosis. Mitochondrial dysfunctionhas been linked to many of the most common neurodegenerative disordersthat are disabling and often fatal diseases. However, the cause ofmitochondrial dysfunction remains largely undefined. It is well knownthat mitochondria interacts physically and functionally with endoplasmicreticulum (ER) via mitochondria-associated membranes (MAM) (Csordas etal., 2006) to influence cellular physiology and viability (Giorgi etal., 2015). Interestingly, the study of MAM has begun to be recognizedas a contributor to neurodegeneration.

Loss of function of the ER protein Wolfram Syndrome 1 (WFS1) leads to aneurodegenerative disease associated with diabetes, optic atrophy anddeafness (Barrett et al., 1995) called Wolfram syndrome. WS wasoriginally described as a mitochondriopathy due to a clinical phenotyperesembling other mitochondrial disorders (Bundey et al., 1993). However,this hypothesis has been challenged by studies showing an ERdistribution of WFS1 (Takeda et al., 2001). Here we report a previouslyunknown function of WFS1 in mitochondrial functionality supporting thenotion that WS is in part a mitochondrial disorder.

Our results reveal that WFS1 is essential to guarantee ER-mitochondriaCa2+ transfer and bioenergetics via its interaction with NeuronalCalcium Sensor 1 (NCS1), a Ca2+ binding protein. This study shows thatWFS1 and NCS1 may participate to the tethering of ER to mitochondria andmay contribute to neurodegeneration.

Results and Discussion

WFS1 Iteracts with and Modulates the Expression of NCS1

To gain insight into how the loss of function of the ER protein WFS1induces mitochondriopathy, we sought to identify protein partners of thecytosolic part of WFS1. We performed a yeast two-hybrid analysis usingresidues 1-311 of murine Wfs1 as bait and identified mouse Ncs1 as aninteracting partner (Data not shown). NCS1 is an EF-hand cytosolicprotein preferentially expressed in neurons (Pongs et al., 1993) andknown to regulate inositol 1,4,5-triphosphate receptor (ITPR) (Nakao etal., 2015; Schlecker et al., 2006; Zhang et al., 2014) and Dopamine D2receptor (Kabbani et al., 2002). To confirm Wfs1/Ncs1 interaction, weperformed a co-immunoprecipitation assay. We showed that Wfs1-myccoimmunoprecipitates with Ncs1-Flag from HEK293T cell lysates (FIG. 1A).This association identifies a novel WFS1 neuronal interacting proteinthat may play an important role in WS.

To investigate NCS1 functions in WS, we took advantage of WFS1-nullpatient fibroblasts. The patients carried two predicted loss-of-functionalleles in WFS1 (V509-Y513del and F882fsX950) and exhibit typical WS. Wefirst examined the expression of WFS1 using quantitative PCR and westernblot. No difference in the mRNA expression level of WFS1 betweencontrols and patients were observed (FIG. 1B). In contrast, the WFS1protein expression level was decreased in patients (Data not shown). Weconfirmed this decrease using immunocytochemistry. These results suggestthat WFS1 loss of function mutations impacted the production/stabilityof the protein rather than the expression/stability of the mRNA. This isin good agreement with previous results showing a decrease stability ofmutant WFS1 protein overexpressed in COS-7 cells (Hofmann and Bauer,2006; Hofmann et al., 2003). Then, we analysed NCS1 mRNA and proteinexpression in patient cells. Similarly, mRNA levels were unchangedbetween controls and patients (Data not shown), whereas NCS1 proteinlevel was decreased by almost 50% in patient fibroblasts (FIG. 1C).Consistent with this observation, knock down of WFS1 using siRNA incontrol fibroblasts decreased NCS1 protein level by 60% (FIG. 1D). Theseresults suggested that WFS1 may regulate NCS1 protein stability.Accordingly, WFS1 has been described as a key regulator of ATF6a levelin stabilizing HRD1 (Fonseca et al., 2010) as well as a regulator ofSERCA expression (Zatyka et al., 2014) especially through the ubiquitinproteasome pathway. Therefore, to determine whether WFS1 affect NCS1expression level involves the proteasome, we used the proteasomeinhibitor MG-132. Interestingly, after 16 h of MG-132 treatment, weobserved a two-fold increase in NCS1 expression in patient fibroblasts(FIG. 1E) whereas in control cells MG-132 was ineffective. Altogether,these results indicate that WFS1 may protect NCS1 from proteasomaldegradation.

WFS1 Interacts with ITPR and Regulates Mitochondrial Ca2+ Uptake

WFS1 is thought to play a role in Ca2+homeostasis by negativelyregulating SERCA turnover and modifying the filling state of the ER Ca2+store (Takei et al., 2006; Zatyka et al., 2014). Moreover, as NCS1modulates the ER Ca2+ release channel, the inositol 1,4,5-triphosphatereceptor (ITPR) (Iketani et al., 2009; Schlecker et al., 2006), wehypothesized that WFS1, NCS1 and ITPR may form a complex to modulateCa2+ flux. To test this hypothesis, we performed co-immunoprecipitationexperiments. We first confirmed the interaction of mouse Ncs1 with ITPRreceptor in HEK293T (Data not shown). Then, we demonstrated theinteraction of mouse Wfs1 with ITPR (Data not shown). These interactionsare consistent with a possible WFS1, ITPR and NCS1 complex formation. Wethen measured the effect of WFS1 loss of function in Ca2+ flux.Cytoplasmic (Fluo-4) and mitochondrial (Rhod-2) [Ca2+] were imaged byconfocal microscopy on fibroblasts after histamine stimulation, known toinduce Ca2+ release from ER stores. Histamine triggered similarIP3-mediated cytosolic [Ca2+] elevations in both control and patientcells, whereas mitochondrial Ca2+ uptake was significantly diminished inpatients (Data not shown). We next measured mitochondrial membranepotential (Δψm) using TMRM dye in control and patient cells. Themitochondrial uncoupler FCCP was applied to dissipate Δψm. Nosignificant difference was observed on FCCP-induced TMRM fluorescencedecreased in both groups indicating a similar Δψm. Therefore, the lackof mitochondrial Ca2+ uptake in patient cells was not due to a smallerdriving force. These results demonstrated that WFS1 physicallyassociates with ITPR and is necessary for ITPR-mediated mitochondrialCa2+ uptake.

Crosstalk between ER and mitochondria is essential for the normalfunctioning of eukaryotic cells. This inter-organelle communicationbetween the ER and mitochondria is crucial for processes such as lipidsynthesis and transport (Vance, 2014), mitochondrial functions (vanVliet et al., 2014), regulation of Ca2+ homeostasis (Patergnani et al.,2011), autophagy (Marchi et al., 2014) and apoptosis (Grimm, 2012). ERdirectly communicates with mitochondria through close contacts referredto as mitochondria-associated membranes (MAMs), which are micro-domainsallowing an efficient Ca2+ transfer between the ER to the mitochondriaand maintaining cellular metabolism and survival. Ca2+ transfer from theER into the mitochondria is facilitated by proteins, which tether thetwo organelles together. For example, the ITPR on the ER interacts withVDAC1 on the outer mitochondrial membrane through the molecularchaperone glucose-regulated protein, GRP75 (Szabadkai et al., 2006).

The ITPR/GRP75/VDAC1 complex also involves the mitochondrial Ca2+uniporter (MCU) on the inner mitochondrial membrane (IMM) (Szabadkai etal., 2006) to allow Ca2+ transfer from the ER to mitochondria.Therefore, we asked if the complex ITPR/GRP75/VDAC1 and MCU wereaffected in Wolfram patient cells. This was first assessed by examiningthe expression of these proteins. There was a marked down-regulation inthe expression of proteins involved in mitochondrial Ca2+ uptake such asVDAC1 in patient fibroblasts. MCU was also decreased in the mostaffected patient P1. No significant differences were found in GRP75 andITPR expression. Thus, we revealed that loss of WFS1 may down regulatesVDAC1 and MCU expression and impairs mitochondrial Ca2+ uptake, possiblythrough down-expression of NCS1.

WFS1 Regulates Mitochondrial Functionality

Reduced mitochondrial Ca2+ uptake might adversely affect variousmetabolic pathways, resulting in altered energy production. We thenevaluated some key enzymes reflecting mitochondrial function, includingCitrate Synthase (CS) and respiratory complexes. CS activity wasdecreased in the mutant cells (25%) when normalized to protein contentsuggesting abnormal mitochondrial integrity. Therefore, we measured theprotein expression level of selected subunits of each complex in themitochondrial electron transport chain. Patient cells presented adecrease in the expression of complex II (SDHA) and complex III (UQCRC2)subunits (FIG. 2A). The expression level of complex I (NDUFA13), complexIV (MTCO1) and complex V (ATP synthase) subunits did not differ betweencontrols and patients (FIG. 2A-2B). These results indicated that theremight be a selective loss of mitochondrial proteins. We wondered whethersuch a decrease could impact mitochondrial bioenergetics. We thusevaluated Oxidative Phosphorylation System (OXPHOS) with oxygenconsumption and enzymatic activity of complexes. Both WFS1 mutant cellsexhibited a significant decrease (20%) of complex II driven respiration(FIG. 2C). Moreover, the patient P1 showed a three-fold diminution ofcomplex I respiration rate (FIG. 2C). To get deeper insights into thecause of the observed decreased mitochondrial respiration in patientfibroblasts, the activities of respiratory chain complexes I (NADHubiquinone reductase), II (succinate ubiquinone reductase) and IV(cytochrome c oxidase) were specifically assessed on cell lysates. Theactivity of complex I was significantly threefold decreased in patientP1 only, whereas the activity of complex II and IV were unchanged in allpatient cells as compared with controls (FIG. 2D). Together, theseresults demonstrated that in Wolfram patients, altered respiration ofcomplex II is linked to the depletion of the complex II amount whereasin the most affected patient P1 the decrease of complex I respirationrate reflected a loss of respiratory capacity. We next assessed celldeath sensitivity in Wolfram patients' cells. Cells proliferation undergalactose was reduced by 30% at 72 h in patient cells and apoptosisinduced by tert-butylhydroperoxyde (tBHP), an activator of mitochondrialapoptosis pathway, was increased in patient cells compared to controls.These results confirm that mitochondrial functionality is impaired inpatient fibroblasts resulting in apoptosis sensitivity of the cells.Yet, no autophagy was observed using biochemical markers as LC3BII/LC3BIratio and Beclin.

NCS1 Regulates VDAC1 Expression and Mitochondrial Respiratory Chain

To elucidate the differential roles of WFS1 and NCS1 on mitochondrialfunctionality, we used short interfering RNAs (siRNAs) to deplete humanfibroblasts of either WFS1 or NCS1. SiRNAs efficiently suppressed WFS1and NCS1 expression 72 h and 96 h post-transfection respectively. Singleknockdown of either WFS1 or NCS1 led to a noticeable decline in bothcomplex I- and complex II-driven respiration as observed in patientfibroblasts (FIG. 3A-3B). Mitochondrial respiration was more affectedwith WFS1 siRNAs due to the partial knockdown of NCS1 associated to thedecrease of WFS1 expression (FIG. 1D). Interestingly, double knockdownof WFS1 and NCS1 induced a decrease of respiration 24 h before NCS1knockdown alone (FIG. 3C). This result suggests that WFS1 and NCS1 sharethe same signaling pathway and that the loss of WFS1 could precipitatethe decrease of NCS1 and the mitochondrial dysfunction associated. Wethen examined whether WFS1 or NCS1 knockdown affect VDAC1 and SDHA(complex II) expression. In NCS1 knockdown cells, VDAC1 and SDHAexpression were significantly reduced compared to controls (FIG. 3D). Incontrast, WFS1 knockdown did not affect VDAC1 and SDHA expression (FIG.3D). This result demonstrated that NCS1 knockdown could down-regulateVDAC1 and be a key determinant for the maintenance of mitochondrialfunction.

Based upon the data provided, we proposed a novel mechanism for WS.WFS1, NCS1 and ITPR may consist of a complex of proteins associated withER-mitochondria contact sites. In healthy cells, WFS1 interacts withNCS1 and prevent its degradation by the proteasome. The complexWFS1/NCS1/ITPR is functional and Ca2+ can transfer from ER tomitochondria properly and activate the tricarboxylic acid cycle (TCAcycle) and mitochondrial respiratory chain. When WFS1 is lost, thecomplex WFS1/NCS1/ITPR is disorganized and NCS1 is partially degraded bythe proteasome. Consequently, altered ER-mitochondrial Ca2+ transferleads to mitochondrial bioenergetic dysfunction that can results in theactivation of cell death.

Importantly, here we demonstrate for the first time an unknown linkbetween WFS1 and NCS1 that is critical for mitochondrial functionality.

Previously, WS studies focused on diabetes because the lack of WFS1causes pancreatic beta cell dysfunction and death (Fonseca et al., 2005;Ishihara et al., 2004; Riggs et al., 2005). However, diabetes isefficiently treated by insulin supplement, whereas neuronal death inbrain, cerebellum and sensory organs is life threatening and unavoidablycauses blindness, deafness and death. The identification of NCS1, a Ca2+binding protein expressed predominantly in neurons, is likely to beinvolved the pathophysiological mechanisms of neuronal defects, possiblydifferent from those in play in the pancreas. It is striking that WSsymptoms resemble those encountered in mitochondrial disorders. Yet,until now, no biochemical evidence supported the notion that part ofthis syndrome was related to mitochondrial dysfunction. The presentfindings of absent mitochondrial Ca2+ uptake and abnormal respiratoryfunctions demonstrate that WS is in part a mitochondrial disorder, inline with the optic atrophy and deafness which are frequently found inmitochondriopathies.

Experimental Procedures

Detailed methods are in Supplemental Experimental Procedures.

Ethical Approval:

The research studies on patient cell lines were approved by the ethicalstandards of the institutional research committee (#11018S) and with the1964 Helsinki declaration.

Cell Cultures

Fibroblasts were cultured from skin biopsies taken after obtaininginformed consent from three controls and two affected patients carryingmutations in WFS1 gene as previously described (Angebault et al., 2011).

siRNA and Transfection

Control fibroblasts were transfected with siRNA directed against WFS1and/or with siRNA directed against NCS1 (Thermo Fisher ScientificBiosciences, ON-TARGET plus Human NCS1 siRNA SMART pool). Transfectionwith an ON-TARGET non targeting pool (siScramble) was used as controls.

Immunofluorescence (IF)

Cells were fixed using 4% PFA and permeabilised using a blockingsolution containing 0.1% Triton X-100 and 5% donkey serum in PBS. Rabbitpolyclonal antibody WFS1 (1:250, Cell Signalling) was incubatedovernight at 4° C.

12

Real-Time RT-PCR

Real-time PCR were performed on total RNA extracted from cells using theRNeasy Mini Kit (Qiagen) and reverse-transcribed with the SuperScriptIII First Strand Kit (Invitrogen) according to the manufacturer'sinstructions.

Enzymatic activities and Oxygen consumption

The activity of the mitochondrial respiratory chain complexes andrespiratory rates were measured on cell homogenates as describedpreviously (Angebault et al., 2011).

Western Blot Analysis

Level of proteins were detected by immunoblot using commerciallyavailable antibodies, revealed using chemiluminescence.

Co-Immunoprecipitation (Co-IP)

For the co-IP studies, HEK293T were transfected with Wfs1-myc,Ncs-1-Flag and Itpr1 using Lipofectamine 2000 according to themanufacturer's instructions.

Yeast Two-Hybrid

Yeast two-hybrid screening was performed by Hybrigenics Services,S.A.S., Paris, France.

Confocal Imaging

Rhod-2 AM (3 μM, Molecular Probes) was used to measure mitochondrialCa2+. To measure cytosolic Ca2+ fibroblasts were loaded with fluo-4 AM(5 μM, Molecular Probes). To measure mitochondrial membrane potential(Δψm), fibroblasts were loaded with 10 nM TMRM (Life technologies).

Statistical Aanalysis

The non-parametric Mann-Whitney U test was used to compare thefibroblasts from WFS1 patients and controls. Differences were consideredsignificant at p<0.05*, p<0.01** and p<0.005***.

REFERENCES

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Example 2: Overexpression of NCS1 Allows Increasing Complex II DrivenRespiration in WS Cells

Fibroblasts of patient's were electropored with Flag alone or Ncs-1-Flagusing basic Fibroblasts Nucleofector kit (Lonza) according to themanufacturer's instructions and processed 24 hours later. Ncs1 wassub-cloned into C-terminal p3XFLAG-CMV between EcoR1 and BamH1.

In mutant cells, NCS1 expression level is decreased by 50% (FIG. 4a ).WFS1 mutant cells exhibited a significant decrease (20%) of complex IIdriven respiration (FIG. 4b ) and a three-fold diminution of complex Irespiration rate (FIG. 4b ). The overexpression of NCS1 in mutant cells(FIG. 4c ) leads to a 30% increased of complex I driven respiration and40% increased of complex II driven respiration compared to Flag alone(FIG. 4d ) 24 h after the transfection.

1. An agonist of NCS1 for use in the treatment of Wolfram Syndrome. 2.The agonist for use according to claim 1, wherein said agoniststimulates the expression of NCS1.
 3. The agonist for use according toclaim 2, wherein said agonist is a NCS1-encoding polynucleotide.
 4. Theagonist for use according to claim 1, wherein said agonist prevents thedegradation of NCS1.
 5. The agonist for use according to claim 4,wherein said agonist is an inhibitor of the proteasome.
 6. The agonistfor use according to claim 4, wherein said agonist is a calpaininhibitor.
 7. A method for assessing the severity of Wolfram syndrome ina subject comprising the step of measuring the level of NCS1 in abiological sample obtained from said subject, wherein the level of NCS1is negatively correlated with the severity of WS.
 8. The methodaccording to claim 7, wherein said biological sample is a fibroblastsample.